Methods for eradicating biofilms from plumbing systems

ABSTRACT

Disclosed are advantageous systems and methods for treating building water systems, especially the interior surfaces of premise plumbing, to remove biofilm and inactivate biofilm-associated pathogens, including protozoa, using disinfectant formulations at concentrations at in excess of those used for drinking water treatment, and further, in co-applying complexing agents to mitigate corrosion of the materials treated; and using these in conjunction with off-gas containment devices that allow flushing of taps without the liberation of toxic fumes.

FIELD

The present disclosure relates to methods for treating plumbing systemsto eradicate, remove and/or disinfect biofilms and biofilm-associatedpathogens using treatment solutions comprising a mixture chlorine andchlorine dioxide.

BACKGROUND

Plumbing associated infections cause tens of thousands of illnesses anddeaths every year. Clinically significant plumbing-associated pathogensinclude Gram-negative environmental bacteria and free-living amoeba(FLA) that can enter plumbing systems in relatively small numbers,reproduce (amplify) to large numbers and release as respirablebio-aerosols from the plumbing into the environment. The onlyplumbing-associated disease requiring notification in the United Statesis Legionnaires' disease, a severe pneumonic infection caused by thebacterium Legionella. Premise plumbing systems are now recognized as theprimary source of Legionnaires' disease. (Yoder et al., 2008) The USCenters for Disease Control and Prevention (CDC) has estimated there areas many as 18,000 cases of Legionnaire's disease annually. The USOccupational Safety and Health Administration (OSHA) has estimated thatLegionnaires' disease results in about 4,000 deaths in the United Stateseach year. Reported outbreaks of Legionnaires' disease have more thandoubled in the past 10 years. Other plumbing-associated pathogens, suchas Pseudomonas and non-tuberculous mycobacteria (NTM), may cause as muchor more disease as Legionella, but lack of required reporting and otherfactors make quantification difficult. The primary disease transmissionvectors for these plumbing associated pathogens are inhalation andaspiration.

Since the early 20th Century, water treatment and disinfection practicesimplemented in the United States and other developed countries havevirtually eliminated incidence of waterborne enteric diseases, such astyphoid and cholera that result from fecal contamination of the publicwater supply. The focus of these historic, successful efforts has beenthe control of “traditional pathogens”, waterborne pathogens of fecalorigin that contaminate the source water and typically do not amplify inthe potable water itself. The primary disease transmission vector forthese traditional pathogens is ingestion.

E. coli is a reference organism of choice in traditional watertreatment; it is widely used as the primary indicator of fecalcontamination. Current data suggest that E. coli is almost exclusivelyderived from the feces of warm-blooded animals; its presence in drinkingwater is considered an indication of substantial post-treatment fecalcontamination or inadequate treatment. E. coli is extremely sensitive tochemical disinfection, such as chlorination. Its presence in a watersample is considered a sure sign of a major deficiency in the treatmentprogram or in the integrity of the distribution system. However, theabsence of E. coli does not, by itself, provide sufficient assurancethat the water is free of microbial contamination.

Constituents of water, pipe deposits and plumbing materials exert aninitial chemical demand on oxidizing disinfectants, such as chlorine.The amount of disinfectant that remains after the initial oxidant demandis satisfied is called the “disinfectant residual”. “Ct”—theconcentration of the disinfectant residual [C] multiplied by the contacttime, “t”—is a key concept used in development of traditionaldisinfection protocols. Ct tables have been developed for each drinkingwater disinfectant for a number of challenge organisms, primarilysuspended (planktonic), traditional (enteric) indicator pathogens suchas E. coli and Giardia.

In general, public drinking water supplies in developed countries aretreated to government standards that make the water safe for intendeduse. In the United States, potable water supplied by community watersystems is treated to National Primary Drinking Water Standards, a setof requirements developed by the United States Environmental ProtectionAgency (USEPA) under authority of the Safe Drinking Water Act (SDWA).Most regulatory mandates regarding drinking water have focused primarilyon the quality of the water at the point it leaves the treatment plant.

It is increasingly recognized that the quality of regulation-compliantdrinking water can deteriorate after it enters the distribution system,the series of pipes that transport water from the treatment plant to thecustomer. In 2006, at the request of USEPA, the National Academy ofSciences published a study by the Water Science Technology Board (WSTB)of the National Research Council (NRC), “Drinking Water DistributionSystems: Assessing and Reducing Risks”. (NRC, 2006) The studyhighlighted the urgent need for new science that will enablecost-effective treatment of the distribution system for protection ofpublic health and minimization of water quality degradation after waterleaves the treatment plant. The distribution system is often categorizedfrom largest to smallest components: transmission (trunk) mains,distribution mains, service lines, and premise plumbing. Typically, thewater treatment utility owns and is responsible for the distributionsystem infrastructure up to the connection to the customer, whichsometimes includes the service line. Almost always, the customer isresponsible for the premise plumbing. The study highlights treatmentchallenges that are unique to premise plumbing.

The term “premise plumbing” refers to the piping within a building orhome that distributes water to the point of use; it also includesequipment used to process the water—that is, to soften, filter, store,heat, and circulate the water before it exits the tap. Premise plumbingsystems are comprised of a wide range of materials including copper,plastics, brass, lead, galvanized iron, and occasionally stainlesssteel. Many of these materials typically are not present in the maindistribution system. Compared to other parts of the water distributionsystem, premise plumbing is characterized by longer water-residencetimes, more stagnation, lower flow conditions, higher surface area tovolume ratio (owing to relatively lengthy sections of small-diameterpipe), lower (if any) disinfectant residual and higher watertemperatures. The distinctive characteristics of premise plumbing createa unique ecological niche and home to a robust microbial ecology.

The microbial colonization of plumbing systems occurs primarily throughthe formation of natural biofilms upon the interior surfaces of theplumbing. (Declerck, 2010; Murga et al., 2001) Biofilms are complexheterogeneous aggregates of microorganisms and exogenous materialsembedded in a highly hydrated matrix commonly referred to asextracellular polymeric substances (EPS). EPS is made up of a variety ofconstituents, including polysaccharides, protein, lipids and nucleicacids. The development, chemical composition, microbial diversity,morphology and activity of biofilms are affected by a number of factors,including water temperature, pH, hardness, disinfection history and thecomposition of the plumbing surface upon which the biofilm forms. Forexample, biofilms that form on copper pipe in a domestic hot watersystem are different from the biofilms that form on the interiorsurfaces of transmission mains, even in the same overall water system.

Biofilm formation on a plumbing surface can be initiated when relativelysmall numbers of environmental microorganisms (such as are typicallyfound in high-quality, regulation-compliant drinking water) enter theplumbing system, attach to the inside surfaces of pipes and equipment,excrete EPS and amplify to very large numbers. Pieces of the biofilm canshed or be dislodged and broadcast as respirable droplets in infectiousbio-aerosols from the plumbing into the environment, for example throughshowerheads, faucet fixtures and ornamental fountains. Infection bythese bio-aerosols is primarily by inhalation and aspiration, andsometimes wound infection.

Clinically important biofilm-associated microorganisms that colonize theinterior surfaces of premise plumbing systems include Gram-negativeenvironmental bacteria, such as Legionella, Acinetobacter,Elizabethkingia (Flavobacterium), Stenotrophomonas, Klebsiella,Pseudomonas and NTM.

Legionella, the most studied plumbing-associated pathogen, survives overa wide range of temperatures. It is acid tolerant to pH 2.0 (Anand etal., 1983) and able to survive temperatures of up to 70° C. (158° F.)(Sheehan et al., 2005). Subject to the availability of necessarynutrients (e.g., iron, L-cysteine), Legionella can grow in water at20-50° C. Legionella proliferate vigorously in water at 32-42° C.(89.6-107.6° F.) with low levels of available nutrients, e.g., inunsterilized tap water (Yee and Wadowsky, 1982), especially inslow-flowing or stagnant water. Legionella is comparatively lesssusceptible to chlorination than E. coli, and reportedly can survivechlorine doses of up to 50 mg/L when contained inside protozoan hosts.

Bacteria and other biofilm-resident microorganisms often arephysiologically different from their free-floating (planktonic)counterparts, and have been shown to be far more resistant totraditional disinfectants, such as chlorine. For example, biofilmbacteria grown on the surfaces of granular activated carbon particles,metal coupons, or glass microscope slides were 150 to more than 3,000times more resistant to hypochlorous acid (free chlorine, pH 7.0) thanwere unattached cells. In contrast, resistance of biofilm bacteria tomonochloramine disinfection ranged from 2- to 100-fold more than that ofunattached cells. (LeChevallier, et al. 1988)

Protozoa play a defining role in the microbial ecology of plumbingsystem associated biofilms. Protozoa graze on biofilm organisms. Anumber of biofilm-associated pathogens (e.g., Legionella, NTM,Pseudomonas) are able to parasitize and replicate within species of FLAcommonly found in drinking water. Once consumed and phagocytized by theprotozoan host, these bacterial pathogens survive, replicate and areeventually dispersed to infect new hosts. While inside the host, thebacteria are protected from environmental stressors, such asdisinfectants and high temperatures. In addition to promoting thebacteria's survival, this process reportedly can result in theup-regulation of the bacteria's virulence genes, and thus directlyaffect their ability to infect humans and cause disease. L. pneumophilahas been shown able to parasitize and multiply in more than twentydifferent protozoan species, including Acanthamoeba, Naegleria, andHartmanella (Donlan et al., 2005; Kuiper et al., 2004). Protozoa havebeen shown to be highly resistant to chlorine and other traditionaldrinking water disinfectants.

The disinfection of public water supplies still relies predominantly onchlorine, but also employs alternative disinfectants chlorine dioxide,monochloramines and ozone to treat water intended for human consumption.(White, G. C. 1999) Chlorine, chlorine dioxide and ozone are used at thetreatment plant, sometimes sequentially, as “primary” disinfectants, toachieve water quality targets in the finished water—that is, at thepoint where the water leaves the plant. Chlorine and monochloramines areadded to the water as “secondary” disinfectants, in order to maintainthe quality of the distributed water all the way to the customer. Ingeneral, the anti-microbial efficacy of each of these disinfectantsincreases with temperature, approximately doubling with each 10° C.increase in water temperature. This finding is consistent with theArrhenius equation, a well-known formula for the temperature dependenceof reaction rates.

Chlorine is the chemical the most frequently used to disinfect publicwater supplies. The pH of the water being treated with chlorine greatlyaffects its disinfection efficacy. Chlorine dissolved in water exists asthree species in equilibrium—chlorine gas (Cl₂), hypochlorite ion (OCl⁻)and hypochlorous acid (HOCl). The ratio of the three components dependson the pH of the water. At pH below 2, chlorine gas becomes significant.When the pH is between 2-7, the equilibrium strongly favors hypochlorousacid, an effective antimicrobial agent. As pH increases above 7,hypochlorous acid dissociates to form hypochlorite ion, which has pooranti-microbial properties. At pH>8, hypochlorite ion dominates.Therefore, when chlorine is used to disinfect water, pH must becontrolled to a lower pH in order to assure that hypochlorous acid, theanti-microbial species, predominates. The amount of chlorine thatremains after the initial oxidant demand of the water is satisfied isknown as the “free residual concentration”. EPA regulations allow a freeresidual chlorine concentration in potable water of up to 4 mg/L.Chlorine at allowable dose levels has proven effective for inactivatinga broad range of traditional (fecal-borne) pathogens in drinking water,Cryptosporidium parvum, an encysted protozoan enteric parasite, is thenotable exception.

Chlorine dioxide is a relatively powerful, fast-acting disinfectant,which inactivates pathogens across a broad pH range, from about pH 5 to9. Chlorine dioxide sometimes is used as an alternative to chlorine forprimary disinfection; however, the ability of chlorine dioxide topersist in the distribution system is unclear. Chlorine dioxidetypically is not used in the United States for secondary disinfection;however, it has been used as a secondary disinfectant in severalEuropean countries including Italy, Germany, France, and Switzerland.

The amount of chlorine dioxide that remains after the initial oxidantdemand of the water is satisfied is known as the “free residualconcentration.” EPA regulations allow a free residual chlorine dioxideconcentration in potable water of up to 0.8 mg/L. Chlorite ion, theEPA-regulated disinfection by-product of chlorine dioxide, has a maximumallowable concentration in potable water of 1.0 mg/L, which effectivelylimits the dose of chlorine dioxide that can be used to treat drinkingwater. The anti-microbial efficacy of chlorine dioxide at pH 5-9 for abroad range of traditional, fecal-borne pathogens in drinking water isroughly comparable or superior to that of chlorine at pH 5-7. Chlorinedioxide is more effective than chlorine against Cryptosporidium.Chlorine dioxide is highly soluble in water but, unlike chlorine,chlorine dioxide does not react with water (hydrolyze); rather, itexists as a dissolved gas. Chlorine dioxide at STP is approximately 10times more soluble in water than is chlorine; the solubility of chlorinedioxide increases as the temperature of the water decreases.

Monochloramine is an oxidant sometimes used as a secondary disinfectant,in order to maintain a relatively weak but persistent disinfectantresidual throughout a distribution system. Monochloramine reacts withorganics at a much slower rate than chlorine; it is therefore is oftenpart of a strategy for minimizing formation of regulated disinfectionby-products associated with chlorine. The anti-microbial efficacy ofmonochloramines at for a broad range of traditional, fecal-bornepathogens in drinking water is far less than that of chlorine orchlorine dioxide. (Van der Wende and Characklis, 1990)

The relative efficacy of chlorine, chlorine dioxide and monochloraminesagainst biofilms and biofilm-associated organisms is different than vs.traditional pathogens. Information on chlorine dioxide efficacy againstbiofilms is inconsistent, though generally seems to be superior to thatof chlorine. Chlorine has limited ability to penetrate biofilms or toinactivate biofilm-resident bacteria, while monochloramine is reportedlyable to penetrate and inactivate organisms within biofilms. M. avium, anNTM species, is more resistant to chlorine than indicator bacteria andsurvives in distribution systems despite ambient chlorine residualconcentrations; most strains appear to be more resistant tomonochloramine compared to free chlorine. All NTM species are believedto be at least 100-fold more resistant to chlorine and otherdisinfectants compared to E. coli (Taylor et al., 2000).

Microbial control treatments applied to plumbing systems fall into twogeneral categories, (1) acute and (2) continuous. Acute treatmentstypically are short-term interventions designed to remediatebio-contamination; continuous treatments typically are part of routineoperations, intended to control bio-contamination. For premise plumbing,conventional acute treatment options have been thermal and chemical.Acute treatment generally been limited to emergency decontamination ofpremise plumbing systems associated with disease outbreaks, owing to theattendant health and safety dangers and damage to the physical plant(e.g., severe corrosion). (White, G. C. 1999)

High-temperature water (e.g., 170° F./77° C.) is sometimes used foracute treatment of domestic hot water systems, in a procedure called“thermal shock” or “super heat and flush”. Thermal shock carriessignificant scalding hazards, is difficult to implement and can causeserious damage to plumbing systems. The high temperatures required tokill plumbing-associated pathogens, such as Legionella, are difficult toachieve and maintain for sufficient time consistently throughout allportions of a premise plumbing system. Even when target temperatures areachieved, thermal shock does not remove established biofilms.

Chemical disinfectants are sometimes used at higher-than-usual doses foracute treatment of pathogen-colonized potable water systems in a processcalled “chemical shock”. The most frequently practiced form of chemicalshock is “hyper-chlorination” using chlorine. The relatively highconcentrations of chlorine employed reportedly cause corrosion, createleaks and otherwise adversely affect plumbing materials. Potable watersystems are likely to be re-colonized within several weeks afterhyper-chlorination. (Williams et al., 2011) Even when pH and chlorineconcentration targets are achieved, hyperchlorination is reportedlyineffective at removing established biofilms. In most hyperchlorinationprotocols, chorine is used at doses sufficient to develop a freechlorine residual of at least 5 mg/L (up to 50 mg/L or more) that ismaintained for up to 24 hours. Because chlorine efficacy is pHdependent, the water must be maintained at less than pH 8 and preferablyless than pH 7.2. Application of such high concentrations of chlorine islikely to corrode pipes and damage plumbing system components,especially at the preferred pH levels where hypochlorous acidpredominates. When flushed through taps, chlorine at the levels used forhyperchlorination can off-gas significantly with release of chlorinefumes substantially above OSHA limits.

A study of acute treatment of a hospital premise plumbing system used ashock dose of 50-80 mg/mL chlorine dioxide applied over an 8-hour periodunder acidic (low pH) conditions; the protocol included flushing of alloutlets at 50-80 mg/mL for approximately 1 hour. Biofilm reportedly wasreduced significantly in the cold and hot taps, but not eliminated;treatment of the showerheads was reportedly unsuccessful, with >3000cfu/ml recovered. (Walker et al., 1997) When flushed through taps,chlorine dioxide at the levels employed in the study can off-gassignificantly with release of chlorine dioxide fumes substantially aboveOSHA limits.

Chlorine, chlorine dioxide and monochloramines are used for continuoustreatment of potable water inside of buildings, especially of domestichot water. Studies with continuous application of chlorine dioxide in ahospital potable water system showed that an extended time (>12 months)was needed to achieve significant reduction in Legionella positivity inhot water system. (Srinivasan, et al., 2003)

The net present replacement value of premise plumbing is in excess of$0.6 trillion (NRC, 2006). Moreover, costs associated with premiseplumbing failures due to corrosion are unpredictable, and include costsof property damage and mold growth. Corrosion of copper pipe, animportant plumbing material, is a function of a number of complexvariables and not fully understood. Chlorine, however, is known to becorrosive to copper pipe. At low pH where hypochlorous acidpredominates, chlorine corrosion can be severe.

Mixtures of chlorine and chlorine dioxide have been reported in theliterature but only in the context of making possible the use of costadvantageous processes for the production of chlorine dioxide or forminimizing the formation of toxic disinfection by-products in bulkwater. For example, Rosenblatt et al. discloses the production of amixture of chlorine and chlorine dioxide using a relatively low costsodium chlorate-based process, with subsequent conversion of thechlorine component to chloramines by the addition of ammonia, oralternatively separation of the chlorine and chlorine dioxide, in orderto remove chlorine and thereby avoid undesirable downstream effects(such as malodors) associated with chlorine-contaminated chlorinedioxide in distribution systems. (Rosenblatt, et al., 1994) Rittman etal. provides for the use of mixtures of chlorine and chlorine dioxide tominimize the formation of regulated disinfection by-products associatedwith chlorine treatment of drinking water in a water treatment plant.(Rittmann et al., 2002) In neither case is the use of a mixture ofchlorine and chlorine dioxide for biofilm treatment taught, nor is theapplication of the mixture to premise plumbing. Katz et. al applied anequal dose of chlorine dioxide and chlorine at pH<7.2, conditions underwhich hypochlorous acid predominates, to disinfect the effluent from amunicipal sewage treatment plant. (Katz et al., 1994) The Katz et al.,results showed that the combination produced relatively-stable residualsof both disinfectants, and reduced the concentration of an undesirabledisinfection byproduct. Katz et. al does not, however, teach use of themixture for biofilm treatment, nor the application of the mixture topremise plumbing. In a study of the inactivation of Legionella in amodel plumbing system, a combination of chlorine and chlorine dioxidedid not show significant synergistic effect. (Zhang, 2007) Norgaarddescribes the use of chlorine dioxide to treat biofilms but neversuggests combining with chlorine. In fact, Norgaard states that biofilmis unaffected by chlorination and points out disadvantages of usingchlorine to treat premise plumbing due to its corrosive properties atlow pH. (Norgaard, 2012).

In order to avoid excessive system noise and the possibility oferosion-corrosion, the generally accepted limits for flow velocities ofdomestic water are 8 feet per second for cold water and 5 feet persecond in hot water, up to approximately 140° F. In systems where watertemperatures routinely exceed 140° F., lower flow velocities such as 2to 3 feet per second should not be exceeded.

SUMMARY

Plumbing-associated disease is a recognized, significant, growing publichealth problem. Effective, practicable means and method for treatingpremise plumbing systems, thereby preventing plumbing-associateddisease, are lacking. Requirements for a viable means and method include(1) removing biofilm and (2) inactivating biofilm-associated pathogens,while (3) minimizing corrosion to the physical plant and (4) mitigatingthe environmental release of toxic chemical fumes. It is a surprisingresult of the methods of the present disclosure that use of a mixturechlorine and chlorine dioxide can remove biofilm and/or inactivatebiofilm-associated pathogens. It is a further surprising result thatsuch mixtures can be effective under conditions that are far lesscorrosive to plumbing system material than the prior artpremise-plumbing treatment methods, which do not use mixtures ofchlorine and chlorine dioxide and which prescribe the use of lower pHwith chlorine-containing solutions. Because the efficacy of chlorinediminishes as pH increases, it is especially surprising the mixtures ofthe present method demonstrates superior biofilm eradication overchlorine or chlorine dioxide alone, even at pH>7.2. As used herein,“biofilm eradication” or “eradicating a biofilm” refers to partially orcompletely destroying a biofilm, which can include partially orcompletely inactivating biofilm-associated pathogens and/or partially orcompletely dislodging and/or removing the biofilm from the surface towhich it is adhered.

In one embodiment, the present disclosure provides a method foreradicating a biofilm from a plumbing system, the method comprisingcontacting the biofilm on interior surfaces of the plumbing system witha treatment solution comprising a mixture of chlorine and chlorinedioxide. In some embodiments, the method can be carried out wherein theplumbing system is a premise plumbing system. In some embodiments, themethod can be used in a plumbing system comprising a building watersystem that is attached to and fed by a premise plumbing system. In someembodiments, the building water system fed by a premise plumbing systemcomprises a cooling tower.

In other embodiments of the method for biofilm eradication from aplumbing system, the treatment solution comprises a mixture of chlorineand chlorine dioxide in a ratio by weight of 50:50. In some embodiments,the treatment solution has a pH of about 6.5 to about 9.0, andoptionally, a pH of about 7.2, about 7.5, about 8.0, or even about 8.5.In some embodiments, the treatment solution has a pH of about 6.5 orgreater, about 7.2 or greater, about 7.5 or greater, about 8.0 or great,or even about 8.5 or greater.

In one aspect of the present invention, methods are disclosed for thetreatment of premise plumbing system wherein the methods comprisecontacting biofilms on the interior surfaces of the plumbing system witha treatment solution, the treatment solution comprising a mixture ofchlorine and chlorine dioxide, and having a pH≥6.5, a pH≥7.2, a pH≥7.5,a pH≥8.0, a pH≥8.5, or a pH≥9.0.

In some embodiments of the method for biofilm eradication, the treatmentsolution concentrations of each of chlorine and chlorine dioxide are atleast 1.5 mg/L (i.e., 3 mg/L total disinfectant). In some embodiments,the treatment solution concentrations of each of chlorine and chlorinedioxide are at least 1.5 mg/L, at least 3.0 mg/L, at least 6.0 mg/L, atleast 12.0 mg/L, or at least 50.0 mg/L. In some embodiments, thetreatment solution, in addition to having a concentration of each ofchlorine and chlorine dioxide described above, optionally also can havea pH of about 6.5, about 7.2, about 7.5, about 8.0, about 8.5, or about9.0.

As used herein, the “treatment solution” is the solution comprising thedesired mix of disinfecting reagents (e.g., a 50:50 chlorine:chlorinedioxide mix) that is present in the plumbing system and therebycontacting the biofilm on the interior surfaces. In some embodiments ofthe method for biofilm eradication, the treatment solution is contactedwith the interior surfaces of the plumbing system by applying a mixtureof chlorine and chlorine dioxide in water into the plumbing system andallowing it to circulate in the premise plumbing system. In someembodiments, the treatment solution can be contacted with the interiorsurfaces of the plumbing system by separately applying chlorine andchlorine dioxide to the plumbing system such that the treatment solutionmixture forms in situ. As described elsewhere herein, residual water canbe present in the plumbing system and thereby can become part of thetreatment solution as a diluent of the component disinfecting reagentsolution(s) added to the system.

In some embodiments, the treatment solution should be prepared such thatthe pH when in the plumbing system is about pH 7.2 or greater. In someembodiments of the method of treatment, the treatment solution has a pHbetween 7.2 and 9.0. In some embodiments the treatment solution has apH>8.0. In some embodiments, the treatment solution has a pH between 8.0and 9.0.

In some embodiments, the methods of treatment for biofilm eradication ofthe present disclosure can be used in plumbing systems wherein theinterior surfaces of the plumbing system are colonized with a biofilmcomprising a microorganism selected from the group consisting ofAcinetobacter, Elizabethkingia (Flavobacterium), Escherichia coli,Klebsiella, Legionella, non-tubercular Mycobacteria (NTM), Pseudomonas,Stenotrophomonas, protozoa, and combinations thereof.

Furthermore, due to the low corrosiveness of the treatment solutionswhen used at high pH (e.g., pH 7.2 and above), in some embodiments themethod of treatment can be carried out wherein the interior surfaces ofthe plumbing system comprise a material selected from the listconsisting of copper, brass, iron, galvanized steel, stainless steel,PVC, HDPE, and combinations thereof.

Generally, in using the methods of the present disclosure, the presenceof any residual water in the plumbing system or additional water addedduring the method can affect the treatment solution disinfectantconcentrations and the pH. Additionally, as described elsewhere herein,residual water in the system can affect the free residual concentrationof the chlorine and chlorine dioxide disinfectants. Accordingly, thepresence and chemical characteristics of water in the system should betaken into consideration when using the methods of treatment disclosedherein.

In some embodiments, the concentration of each of the components of thetreatment solution can be at or below the levels permitted in drinkingwater or higher than the levels permitted in drinking water. Thus, insome embodiments of the method of treatment, the chlorine-to-chlorinedioxide ratio by-weight in the treatment solution can be from 80:20 to20:80, and in some preferred embodiments, can be a ratio by-weight of50:50. In general, the higher the concentration of the treatmentsolution, the lower the amount of contact time required to treat thepremise plumbing.

In some embodiments of the methods of treating premise plumbingdisclosed herein, the chlorine and chlorine dioxide concentration levelsin the treatment solution are 0.8 mg/L or less residual chlorine dioxideand 0.4 mg/L or less residual chlorine. In other embodiments, thechlorine and chlorine dioxide concentration levels in the treatmentsolution are greater than 0.8 mg/L residual chlorine dioxide and greaterthan 0.4 mg/L residual chlorine.

In another aspect of the present invention, the treatment solution iseffective across a broad range of temperatures, including the full rangeof temperatures characteristic of domestic water (0-60° C.; 32-140° F.).Accordingly, the methods of treating premise plumbing disclosed hereincan be carried out across a wide range of temperatures, and even at coldwater temperatures. In some embodiments, the temperature of thetreatment solution is between 55 to 80° C. In other embodiments, thetemperature of the treatment solution is between 20 to 55° C., and insome embodiments, the temperature of the treatment solution is between 0to 20° C. Indeed, application at cold water temperatures offers theadvantage of increased chlorine dioxide solubility. Also, coldertreatment solution can be circulated faster than warmer treatmentsolution and offers more latitude in achieving desired turbulent flow,which can enhance biofilm removal by virtue of increased sheer forcesand better mixing at the treatment solution:biofilm interface.Application of the treatment solution at warmer temperatures offersadditional efficacy due to increased rates of reaction, but increasesthe release of chemical fumes (off gassing) and the rate at which thetreatment solution reacts with organic constituents of the water andwith plumbing materials. An additional consideration is that aqueoussolutions at temperatures>43.3° C. (110° F.) can scald.

In another aspect of the present invention, the application of thetreatment solution can be advantageously carried out by circulatingthrough the premise plumbing at a flow rate a Reynolds value of at least4,000.

When in contact with the plumbing system surfaces, the treatmentsolution can be at temperatures between 0 and 80° C. (32-176° F.) and pHvalues ranging between pH 6.5-9, preferably pH 7.2-8. The treatmentsolution may also include a complexing agent, such as sodium silicate,to further mitigate corrosion, especially when the treatment solution isapplied at higher temperatures and higher concentration. In anotheraspect, the present invention is directed to methods for containinggases at open taps during acute chemical treatment of premise plumbingsystems when the treatment solution is being flushed through taps. Themethod comprises attaching a conduit to an outlet (e.g., tap), whereinthe conduit walls are partially or fully impermeable to chlorine andchlorine dioxide gas, thereby forming a partial or complete barrier tothe transmission of these gas vapors. The conduit may be a pipe, tube,hose, channel or the like. The opposing end of the conduit is directedtoward a physical or chemical sink that prevents liberation of chemicalfumes into the environment; examples of embodiments include, withoutlimitation, the opposing end of the conduit being (a) terminated justabove a drain, with a small air gap; (b) fitted into a drain or (c)attached to the tapered end of a funnel wherein contact between theconduit and the funnel is sealed, and the funnel is a barrier tochlorine dioxide gas and is (i) fitted to a drain, (ii) secured to adrain, (iii) sealed to a drain, or (iv) in close proximity to a drain.In another example, the opposing end of the conduit also may be passedthrough a plug fitted in the drain, wherein the plug occludes the vaporpath from the drain. In one variation, the plug may be a sponge soakedin a scrubber solution, such as an aqueous solution containing sodiumthiosulfate (a reducing agent) that inactivates the chlorine andchlorine dioxide fumes. The sponge is fitted into a drain and maycontain a chemical scrubber. The water source is a (a) tap, (b) showerhead, or (c) fixture. The conduit may contain (a) check valves, (b)remotely actuated valves, (c) temperature sensors, (d) pH sensors, (e)chemical sensors, (f) devices for data acquisition, (g) devices for datastorage, and/or (h) devices for data transmission. The treatmentsolution is passed through the conduit while maintaining isolationbetween potable water and waste water systems, as may be required byplumbing codes.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A is a schematic illustration of a conduit between the tap and thedrain. The upper dashed line indicates the level of the tap and thelower dashed line indicates the level of the drain. The arrow indicatesthe direction of water flow, which is from the tap to the drain.

FIG. 1B is a schematic illustration of a conduit between the tap and thedrain. The conduit contains a check valve. The upper dashed lineindicates the level of the tap and the lower dashed line indicates thelevel of the drain. The arrow indicates the direction of water flow,which is from the tap to the drain.

FIG. 1C is a schematic illustration of a conduit between the tap and thedrain. The conduit is attached to the inverted end of a funnel. The wideend of the funnel is at the level of the drain. The upper dashed lineindicates the level of the tap and the lower dashed line indicates thelevel of the drain. The arrow indicates the direction of water flow,which is from the tap to the drain.

FIG. 1D is a schematic illustration of a conduit between the tap and thedrain. The conduit is fitted through a sponge and the sponge is at thelevel of the drain. The upper dashed line indicates the level of the tapand the lower dashed line indicates the level of the drain. The arrowindicates the direction of water flow, which is from the tap to thedrain.

FIG. 1E illustrates a conduit attached to the tap of a sink and leadinginto the drain of the sink.

DETAILED DESCRIPTION

For the descriptions herein and the appended claims, the singular forms“a”, and “an” include plural referents unless the context clearlyindicates otherwise. The use of “comprise,” “comprises,” “comprising”“include,” “includes,” and “including” are interchangeable and notintended to be limiting. It is to be further understood that wheredescriptions of various embodiments use the term “comprising,” thoseskilled in the art would understand that in some specific instances, anembodiment can be alternatively described using language “consistingessentially of” or “consisting of.” The technical and scientific termsused in herein will have the meanings commonly understood by one ofordinary skill in the art, unless specifically defined otherwise.

Where a range of values is provided, unless the context clearly dictatesotherwise, it is understood that each intervening integer of the value,and each tenth of each intervening integer of the value, unless thecontext clearly dictates otherwise, between the upper and lower limit ofthat range, and any other stated or intervening value in that statedrange, is encompassed within the invention. The upper and lower limitsof these smaller ranges may independently be included in the smallerranges, and are also encompassed within the invention, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding (i) either or (ii)both of those included limits are also included in the invention. Forexample “1 to 50” includes “2 to 25”, “5 to 20”, “25 to 50”, “1 to 10”,etc.

It is to be understood that both the foregoing general summary and thefollowing detailed description, including the drawings and examples, areexemplary and explanatory only and are not restrictive of the inventionsof this disclosure.

Mixtures of chlorine and chlorine dioxide in a treatment solution aresurprisingly effective for eradicating biofilms on interior surfaces ofplumbing systems via killing (or inactivating) of biofilm-associatedpathogens and dislodging and/or removing the biofilms, even at high pH(e.g., pH>7.2) which results in minimal plumbing system corrosion. Thisdiscovery holds true for treatment solutions of chlorine and chlorinedioxide where the concentrations of these disinfectants are at therelatively-low levels permitted in drinking water (e.g., 0.8 mg/Lchlorine dioxide; 4 mg/L chlorine). The method and treatment solutionsis also effective to eradicate biofilms at much higher concentrations(e.g., 25 mg/L chlorine dioxide; 25 mg/L chlorine). Additionally, it issurprising that this discovery holds true at pH values much greater than7.2, (e.g., pH 8.5), and even in domestic cold water (e.g., 0-20°C./32-68° F.).

The co-application of a complexing agent, such as sodium silicate, withtreatment solution can provide enhanced protection of the metalliccomponents of premise plumbing materials from corrosion, especially whenthe treatment solution is applied at higher concentrations and highertemperatures.

The penetration of biofilm on the surfaces of pipes is benefited byincreased flow and most markedly by turbulent flow at thebiofilm:treatment solution interface. Without being limited by theory,we believe this is likely is due to enhanced contact between thetreatment solution and the surface being treated by eliminating aboundary layer associated with laminar flow and/or stagnant contact. Thedegree of turbulence that can be achieved is a function of flow rate andpipe diameter; for example, a 2-inch diameter pipe requires a flow rateof approximately 2 feet per seconds to achieve turbulent flow (Reynoldsnumber˜4,000).

The removal of biofilm on the treated surfaces is also benefited byincreased flow. Without being limited by theory, we believe this isbecause of increased shear forces that have a scouring effect on thebiofilm.

Based on these discoveries, certain embodiments of the invention providenovel means and methods for treatment of premise plumbing systems.

For treatment of premise plumbing systems, it is desirable to removesurface-attached biofilm and to kill biofilm-associated pathogens, suchas bacteria, viruses and protozoa, without causing significant physicaldamage to pipes and other premise plumbing system components and withoutenvironmental release of noxious chemical fumes. Removingsurface-attached biofilm and killing biofilm-associated pathogenswithout damaging copper pipes and other system components can be met byflushing the premise plumbing system with a mixture of chlorine andchlorine dioxide in aqueous solution (treatment solution). The treatmentsolution can comprise a mixture of chlorine and chlorine dioxide at aratio of 80:20 to 20:80 (weight basis) at a total concentration of up to200 mg/L for up to 24 hours. The treatment described herein may beadvantageously practiced at treatment solution pH values greater thanneutral, especially pH>7.2, at typical cold water temperatures (e.g.,0-20° C.; 32-68° F.) up to temperatures at which scalding becomes a risk(43.3° C./110° F.). Co-treating with a complexing agent, such as sodiumsilicate, can further enhance compatibility of the treatment solutionwith plumbing system materials, especially metals such as copper andbrass.

Environmental release of noxious chemical fumes at the tap can beavoided by utilizing a gas-containment device, such as a hose thatcontains chlorine and chlorine dioxide vapors, which hose is attached toa tap and terminates near or is attached to a drain or chemical scrubberand provides containment of chemical fumes and directs the flow oftreatment chemicals from the tap down a drain or into a chemicalscrubber. The gas-containment device can be configured in many ways, andcan incorporate advantageous features such as check valves, remotelyactuated valves, and sensors for temperature, pH and disinfectantconcentration, as well as data acquisition, storage and transmissionmeans. The gas-containment device can be used in conjunction withflushing the treatment solution, or with other volatile treatmentchemicals, such as hypochlorite (bleach) or chlorine dioxide, that maybe used to flush plumbing systems.

In certain plumbing systems with surfaces coated by a mixture of limescale, iron sediment and biofilm, such as those that receive water withhigh mineral content (hard water), treatment can be carried out by firstapplying a low pH treatment to dissolve the limescale, and iron,preferably in conjunction with a complexing agent, such as sodiumsilicate. The first step is followed by utilizing the treatment solutiondescribed herein at higher pH (e.g., >7.2) to remove the biofilm. Thissequence can be repeated, if necessary, until the limescale, ironsediment and biofilm have been removed as determined by sampling orvisual inspection.

EXAMPLES

Various features and embodiments of the disclosure are illustrated inthe following representative examples, which are intended to beillustrative, and not limiting. Those skilled in the art will readilyappreciate that the specific examples are only illustrative of theinvention as described more fully in the claims which follow thereafter.Every embodiment and feature described in the application should beunderstood to be interchangeable and combinable with every embodimentcontained within.

Example 1 Eradication of Pseudomonas Biofilms Using Solutions ofChlorine, Chlorine Dioxide, and a Mixture of Both

This example illustrates the use of a mixture of chlorine and chlorinedioxide for removing biofilms of Pseudomonas aeruginosa. These resultsshow the efficacy of this treatment solution even at pH 6.5, and thesurprising advantage of such mixtures at pH 7.5 relative to treatmentsolutions comprising either chlorine or chlorine dioxide alone for thetreatment of biofilms.

Overview of Testing Method

Pseudomonas aeruginosa biofilms are challenged with the reagentschlorine dioxide (“CD”), Chlorine (sodium hypochlorite; “Bleach”) or amix of both reagents in equivalent volume proportions (“Mix”), atapproximately pH 6.5 and pH 7.5 to assess their ability for biofilminactivation, as measured by a Minimal Biofilm Eradication Concentration(MBEC) assay. This MBEC assay measures the ability of a treatmentsolution to eliminate bacteria from already grown biofilms by killingthe microbial cells and/or dislodging the biofilm.

The MBEC assay protocol used is an adaptation of the ASTM-standardizedMBEC method. Briefly, biofilms are grown in a Calgary biofilm device,which is a 96 well plate with a lid that when in place has 96 individualpegs that protrude into the wells of the plate (see e.g., Ceri et al.,1999). The bacterial cells in the wells form biofilms on the submergedpegs. After a 20 h growth period, these biofilms formed on the pegs arechallenged by placing the pegged lid on a 96-well plate of serialdilutions of the disinfectants to be tested (e.g., the CD, Bleach, orMix), along with the required controls. All challenges were performed ina Biosafety Level 2 cabinet with a challenge exposure time of 30 minutesat a temperature of 22-25° C. In the case of CD, products are preparedfresh from highly concentrated stock solution minutes before thechallenge. Reagent pH and concentration stability are tested for the CDworking stock solutions, using either water or a borate-boric acidbuffer as solvents. The concentration ranges for disinfectant reagentsare from approximately 3 ppm to approximately 50 ppm.

After the challenge, the pegged lid is transferred to a recovery platecontaining a sodium bisulfite solution. Sonication of the recovery platewith the pegged lid induces detachment of the biofilm from the pegs intothe wells of the recovery. Viable bacterial cell counts from each pegare determined by culturing the recovery plate. The extended MBEC assayprotocol and methods of preparing the disinfectant reagent stocksolutions used in the challenge are included in the Materials andMethods section provided below.

Results

Results for CD, Bleach and the Mix challenges can be seen in Tables 1, 2and 3, respectively. Bleach challenge shows a pH-dependent effect forbiofilm reduction, consistent with the literature, except for anomalousresults at ˜3 ppm. When Mix (Bleach and CD) was used (Table 3), at bothpH 6.5 and pH 7.5 efficacy against biofilms was superior to the efficacyof either of these disinfectant reagents alone. This is most clearlydemonstrated by comparing the dose of the Mix of 6.25 ppm of eachcomponent (12.5 ppm total) vs. 12.5 ppm of either componentindividually.

TABLE 1 Results of challenge of P. aeruginosa biofilms with CD. pH 6.5pH 7.5 Concentration Log₁₀ reduction Concentration Log₁₀ reduction (ppm)(CFU/mm²) (ppm) (CFU/mm²⁾ 50.0 Total inhibition 50.0 total inhibition12.5 2.34 12.5 2.49 6.25 2.56 6.25 2.38 3.12 0.82 3.12 1.63 Log₁₀reduction = logarithm of the number of Colony Forming Units (CFU) permm² after culturing the ultrasound-recovered biofilm.

TABLE 2 Results of challenge of P. aeruginosa biofilms with Bleach. pH6.5 pH 7.5 Concentration Log₁₀ reduction Concentration Log₁₀ reduction(ppm) (CFU/mm²⁾ (ppm) (CFU/mm²⁾ 50.5 Total inhibition 50.5 Totalinhibition 12.6 Total inhibition 12.6 3.03 6.31 Total inhibition 6.311.91 3.15 0.16 3.15 0.96 Log₁₀ reduction = logarithm of the number ofColony Forming Units (CFU) per mm² after culturing theultrasound-recovered biofilm.

TABLE 3 Results of challenge of P. aeruginosa biofilms with a Mix (CDand Bleach). pH 6.5 pH 7.5 Concentration Log₁₀ reduction ConcentrationLog₁₀ reduction (ppm) (CFU/mm²⁾ (ppm) (CFU/mm²⁾ 50.0 Total inhibition50.0 Total inhibition 12.5 Total inhibition 12.5 Total inhibition 6.253.08 6.25 Total inhibition 3.13 2.18 3.13 1.99 Log₁₀ reduction =logarithm of the number of Colony Forming Units (CFU) per mm² afterculturing the ultrasound-recovered biofilm.

Detailed Materials & Methods

Preparation of Concentrated CD Solution:

If available, pour approximately 50 mL of a CD solution in two 1 L glasscontainers that can be airtight sealed. Otherwise, use commercial bleachto treat this glassware. Make the liquid moisten all of the innersurface of the flasks, and let them stand in the dark at roomtemperature overnight. Add 2 L of sterile, distilled water to a pouchcontaining the CD-producing reagents. Cap the pouch, and mix byinverting it several times. Let stand for 2 hours at room temperature.Rinse with sterile, distilled water the glassware from point 2, andcover them with tin foil. Transfer the Concentrated CD solution to theglass bottles, so that the air space on top of the solution is asminimum as possible. Store the solution at 4° C. until needed. Prepare30 mL of 1:500 to 1:1000 dilutions. Transfer 10 mL aliquots to 20 mLbottles of the quantitation system (CD colorimeter). Use aluminum foilto cover the bottles. Add 3 drops of glycine to each bottle, cap it, andmix. Add the content of one bag of DPD to each bottle with a CDsolution. Mix by gently inverting the tubes. Use a bottle with water toblank the colorimeter (make sure you are using the CD, and not thechlorine (Bleach) colorimeter), and then measure the concentration. Ifreading is >2.5, prepare a higher secondary dilution (1:1500) and repeatthe process. Average the readings and multiply by the dilution factor todetermine the power (in ppm) of the concentrated solution. Label thebottles from with date and concentration.

Preparation of Working CD Stock Solution:

Fill a 50 mL polypropylene tube with the concentrated solution, andcover it with aluminum foil. Let the solution reach room temperaturewhile protected from light. In a fresh 50 mL polypropylene tube, mix theconcentrated solution with sterile, distilled water, so that it isdiluted to the working concentration (e.g., 100 ppm), in a final volumeof 50 mL. Close tightly the tube, and mix gently by inversion. Add 0.1MNaOH in 10 μL increments, mix and measure pH. Repeat this step until thedesired pH is reached. Also, estimate pH using strips of pH indicatorpaper. Prepare 1:200 dilutions (30 mL), and verify the workingconcentration using the colorimeter (as described in making concentratedCD solutions). Use concentration estimation strips for double checking.Submerge the quantitation end of the strip in the solution for 2seconds. Holding the colorimetric coupon upright, wait for 10 seconds,and then compare its color with the reference pallet. Label the tube,and store it at 4° C.

Preparation of 1× Borate-Boric Acid Buffer Working CD Stock Solutions:

In a fresh 50 mL polypropylene tube, place concentrated CD solution sothat the final concentration after dilution to a volume of 50 mL will bethe working concentration (e.g. 100 ppm). Add 1× borate buffer to reach40-45 mL. Close tightly the tube, and mix gently by inversion. MeasurepH, and add 0.5M boric acid in 1 mL increments (or less if needed) untilthe desired pH is reached. Complete to 50 mL volume with sterile,distilled water. Determine final concentration as described above for CDstock solution.

Stability Testing of Working CD Stock Solutions:

The method for pH and concentration stability testing consisted ofdiluting CD from a concentrated stock solution (approximately 590 ppm)to the working stock solution concentration (100 ppm) using water andadding 0.1M NaOH in small amounts until the desired pH was reached.Alternatively, the reagent was diluted to its working concentration in acombination of 1× borate buffer (measured pH 8.8) and 0.5M boric acid(measured pH 4.14). The desired pH levels were reached by adjusting theproportion of boric acid in the solution.

Initial Concentration Estimation of Starting Bleach Solution:

Bleach can come at concentration spanning 1 to 8% (i.e., 10000 to 80000ppm). The following initial concentration estimation method is used todetermine this starting concentration. In a new polypropylene tube,prepare 15 mL of a 1:500 dilution in Ultra-Pure Water (UPW). Place 10 mLof UPW in the 20 mL bottles of the quantitation system (Cl₂colorimeter). Put aside one of the bottles to use it as blank. Replaceeither 100 or 50 μL of UPW with the 1:500 dilution from step (1) (i.e.,make 1:100 or 1:200 further dilutions, this will make final dilutions of1:50000 and 1:100000 respectively). Add the content of one bag of DPDreagent to each bottle with a CD solution. Mix by gently inverting thetubes. Blank the colorimeter (make sure you are using the chlorine, andnot the CD colorimeter), and then measure the concentration. If readingis >2.5, prepare a higher secondary dilution (for example 1:1500) andrepeat the process. Average the readings and multiply by the dilutionfactor to determine the power (in ppm) of the concentrated solution.Label the original bottle with date and estimated concentration.

Preparation of Bleach Working Stock Solutions:

Fill a 50 mL polypropylene tube with the concentrated Bleach solution.Let the solution reach room temperature. In a fresh 50 mL polypropylenetube, mix the concentrated solution with sterile, distilled water, sothat it is diluted to the working concentration (e.g. 100 ppm), in afinal volume of 50 mL. Close tightly the tube, and mix gently byinversion. Add 1N HCl in 10 μL increments, mix as in (9.2) and measurepH. Repeat this step until the desired pH is reached. Also, estimate pHusing strips of pH indicator paper. Prepare 1:200 or 1:100 dilutions,and verify the working concentration as before (steps 2-6). Label thetube, and store it at 4° C. until use.

MBEC Assay Protocol

The following equipment, reagents, and methods are used to carry out theMBEC assay protocol for testing disinfectant efficacy of chlorinedioxide (CD), chlorine (Bleach) and a mixture of chlorine dioxide andbleach (Mix) against Pseudomonas aeruginosa biofilm.

A. Assay Method: All steps should be performed using aseptic techniquesand in an aseptic environment.

1. Bacterial culture (2 days before the MBEC assay).

-   -   1.1. Thaw an aliquot of a working stock of Pseudomonas        aeruginosa (ATCC 27835) and use it to streak plate on tryptic        soy agar (“TSA”) prepared according to manufacturer's        directions.    -   1.2. Incubate at 35° C. for 16-18 h.    -   1.3. Pick an isolated colony and with it inoculate 200 mL of        sterile tryptic soy broth (“TSB”) prepared according to        manufacturer's directions.    -   1.4. Incubate at 35° C. 150 rpm for 16 to 18 h. Viable bacterial        density should be 10⁸ CFU/mL or higher and may be checked by        serial dilution and plating.    -   1.5. Prepare a 25 mL 1:1000 dilution in TSB, to adjust cell        density to approximately 10⁵ CFU/mL. Vortex the diluted sample        for approximately 10 s.    -   1.6. Perform seven 10-fold serial dilutions from step (1.5) in        triplicate.    -   1.7. Spot plate 20 μL of the serial dilutions from 10° to 10⁻⁷        on a series of TSA plates. Label plates and incubate them at        35° C. for 16-18 h.

2. Growth of biofilm

-   -   2.1. Open a package containing a new MBEC device.    -   2.2. Transfer 25 mL of the inoculum prepared in (1.5) into a        sterile reagent reservoir.    -   2.3. Add 150 μL to each well of the 96-well plate packaged with        the MBEC device, excluding columns 9 to 11 and A12, B12 and C12.    -   2.4. Place the peg lid onto the microplate, making sure that the        orientation of the wells matches that of the peg lid (i.e., peg        A1 must be inserted into A1 well).    -   2.5. Using the orbital shaker and humidified incubator, keep the        device at 33-37° C.    -   2.6. For best biofilm quantitation, the replicate MBEC devices        should be prepared according to section 7.

3. Biofilm growth check

-   -   3.1. Using sterile (flamed) pliers, grab peg D12 close to the        lid to avoid contact with the biofilm. Break off the peg and        place it in a sterile microfuge tube containing 1.0 mL of        buffered water (“buffered water”=0.0425 g KH₂PO₄/L distilled        water, filter-sterilized and 0.405 g MgCl.6H₂O/L distiller        water; filter-sterilized, as according to ASTM Method 9050        C.1.a),    -   3.2. Repeat step (3.1) with wells E12 to H12 into respective        microfuge tubes.    -   3.3. Place the stainless steel insert tray into the sonicator.        Place the tubes from 3.1 and 3.2 into the tray and sonicate on        high for 25-35 min    -   3.4. Make 1.0 mL 10-fold serial dilutions in buffered water and        spot plate on TSA. Incubate at 35° C. for 16-18 h.

4. Preparation of challenge plate

-   -   4.1. Use a sterile 96-well, two corners plate, to prepare the        challenge plate according the challenge plate set-up map shown        below.

1 2 3 4 5 6 7 8 9 10 11 12 A 100 100 100 100 100 50:N N UC SC B 50 50 5050 50 50:N N UC SC C 25 25 25 25 25 50:N N UC SC D 12.5 12.5 12.5 12.512.5 50:N N UC BGC E 6.25 6.25 6.25 6.25 6.25 50:N N UC BGC F 3.13 3.133.13 3.13 3.13 50:N N UC BGC G 1.56 1.56 1.56 1.56 1.56 50:N N UC BGC H0.78 0.78 0.78 0.78 0.78 50:N N UC BGC

-   -   4.2. Prepare 20 mL of the desired disinfectant stock solution.    -   4.3. Add 200 μL of sterile TSB to well A12 of the challenge        plate. This will be the sterility control (SC).    -   4.4. Add 200 μL of sterile neutralizer to column 7 and well B12.        These will be the neutralizer toxicity control (N) and sterility        control.    -   4.5. Add 100 μL of sterile neutralizer to column 6, followed by        100 μL of disinfectant. This will be the neutralizer        effectiveness control.    -   4.6. Add 200 μL of buffered water to column 8 and well Cl₂.        These will be untreated control (UC) and buffered water        sterility control.    -   4.7. Add 100 μL of buffered water to columns 1 through 5 (rows B        through H).    -   4.8. Add 200 μL of stock disinfectant to columns 1 through 5        (row A).    -   4.9. Add 100 μL of the disinfectant stock solution to columns 1        through 5 (rows B and C).    -   4.10. Use a multichannel micropipette to mix the contents of        columns 1 through 5 (row C) by pipetting up and down. Keep the        tips in the micropipette for the next step.    -   4.11. Transfer 100 μL from the wells in row C to the        corresponding wells in row D. discard the tips.    -   4.12. Using fresh tips, mix by pipetting the contents in row D,        columns 1 through 5.    -   4.13. Transfer 100 μL from row D to row E. Discard the tips        after each transfer and mix with fresh ones.    -   4.14. Repeat the process down the length of the plate until row        H.    -   4.15. Discard 100 μL, from row H, columns 1 to 5.    -   4.16. Add 100 μL, of buffered water to rows C through H of        columns 1 through 5.

5. Disinfectant challenge of biofilm

-   -   5.1. Prepare a rinse plate by adding 200 μL, of buffered water        to each well of a new 96-well, 2-corners plate.    -   5.2. Prepare recovery plate by adding 200 μL, of neutralizer to        each well of a new 96-well, 2-corners plate.    -   5.3. Rinse the planktonic bacteria from the biofilm that formed        on the lid of the MBEC device by setting the lid into the rinse        plate for 10 s.    -   5.4. Transfer the MBEC lid to the challenge plate and incubate        at room temperature during the contact time recommended by the        manufacturer.    -   5.5. After the contact time, transfer the MBEC lid to the        recovery plate containing the neutralizer.

6. Biofilm growth quantitation (from replicate biofilm plate)

-   -   6.1. Prepare a staining plate by adding 200 μL, of 0.1% crystal        violet solution into columns 1 through 8 and column 12 of a        fresh 96-well, 2 corners plate.    -   6.2. Transfer the pegged lid of the replicate recovery plate to        the staining plate.    -   6.3. Incubate at room temperature for 30 minutes.    -   6.4. Prepare two rinse plates by adding 200 μL, of ultrapure        water into columns 1 through 8 and column 12 of two 96-well, 2        corners plates.    -   6.5. Transfer the lid to the first rinse plate and let it settle        for 10 s, to remove excess of stain and planktonic bacteria.        Transfer to the second rinse plate and repeat.    -   6.6. Let the pegged lid air-dry, upside down, for 30 minutes.    -   6.7. Add 150 μL, of 95% ethanol to a new 96-wells, 2 corners        plate, in columns 1 through 8, and column 12.    -   6.8. Once dry, place the pegged lid into the ethanol plate and        incubate for 10 minutes.    -   6.9. Remove the pegged lid and discard it, along with the        staining and rinse plates used in this section.    -   6.10. Transfer 100 μL, of each well from the plate in (6.8) into        the corresponding well of a fresh 96-wells (ONE CORNER) plate.    -   6.11. Use a plate reader to determine absorbance at 600 nm.

7. Quantitative determination of the MBEC

-   -   7.1. Place the recovery plate with the pegged lid (from step        6.5) in the stainless steel tray, and the tray, in the        sonicator. Sonicate on high for 25 to 35 min to remove and        disaggregate the biofilm.    -   7.2. Eight sterile 96-well, ONE CORNER plates are used for this        step (columns 1 through 8 only).        -   7.2.1. Add 180 μL of buffered water to rows B through H in            all 8 plates.        -   7.2.2. Following sonication and using multichannel            micropipette, transfer 100 μL from each well of row A of the            recovery plate to row A of a sterile plate prepared in            7.2.1.        -   7.2.3. Transfer 100 μL from each well of row B of the            recovery plate to row A of a second sterile plate prepared            in 7.2.1.        -   7.2.4. Repeat for rows C through H of the recovery plate.        -   7.2.5. Serially dilute with a multichannel pipette (10° to            10⁻⁷) by transferring 20 μL down each of the 8 rows for each            plate.    -   7.3. Spot plate the dilution series from each of the eight        microtiter plates on TSA for viable cell counts. Use one square        TSA plate per microtiter plate. Using a multichannel pipette,        remove 5 μL from each well and dispense on TSA plate.    -   7.4. Incubate the TSA plates at 33-37° C. during 18 to 20 h and        enumerate colonies.    -   7.5. Discard the pegged MBEC lid and 96 plates used to create        the serial dilutions.

8. Qualitative determination of the MBEC

-   -   8.1. Add 100 μL of sterile TSB to each well of the recovery        plate.    -   8.2. Cover recovery plate with a new sterile, non-pegged lid and        place in a humidified incubator at 33-37° C. for 24 h.

9. Data Analysis

-   -   9.1. Quantitative MBEC results using Login reduction:        -   9.1.1. Count the 5 μL spots on each of the 8 spot plates            where individual colonies are visibly distinct from each            other within the plated spot. Record the column (1-8) and            dilution row (10⁰ to 10⁷) in which each spot is located.        -   9.1.2. Calculate the log₁₀ density for each peg as follows:            Log₁₀(CFU/mm²)=Log₁₀[(X/B)(V/A)(D)]        -   where:            -   X=CFU counted in the spot,            -   B=volume plated (0.01 mL),            -   V=well volume (0.20 mL),            -   A=peg surface area (46.63 mm2), and            -   D=dilution        -   9.1.3. Average the counts from columns 1 through 5 spot            plated for Row A to determine the mean log₁₀ density for the            undiluted disinfectant.        -   9.1.4. Average the counts from columns 1 through 5 spot            plated for Row B to determine the mean log₁₀ density for the            50% disinfectant. Repeat calculation for the remaining rows            (C—H).        -   9.1.5. Average the counts from column 6, Rows A through H to            determine the mean log₁₀ density for the neutralizer            effectiveness control according to the procedure described            in TSA Test Method E1054.        -   9.1.6. Average the counts from column 7, Rows A through H to            determine the mean log₁₀ density for the neutralizer            toxicity control.        -   9.1.7. Average the counts from column 8, Rows A through H            determine the mean log₁₀ density for the untreated control.        -   9.1.8. Calculate the log₁₀ reduction for each disinfectant            concentration as follows:            Reduction=Mean Log₁₀ Untreated Control Pegs−Mean Log₁₀            Treated Pegs    -   9.2. Qualitative MBEC Results are determined following the 24 h        incubation of the recovery plates by visual scoring (+/−        growth). To determine the minimum biofilm eradication        concentration (MBEC) values, check for turbidity (visually) in        the wells of the recovery plate. Alternatively, use a microtiter        plate reader to obtain optical density measurements at 650 nm        (OD₆₅₀). Clear wells (OD₆₅₀=0.1) are evidence of biofilm        eradication. The MBEC is defined as the minimum concentration of        disinfectant that eradicates the biofilm. This is the lowest        concentration in which there is no growth observed in the        majority of the five wells.

Example 2 Biofilm Eradication in a Recirculating Domestic Hot WaterSystem Using a Treatment Solution Mixture of Chlorine and ChlorineDioxide

This example illustrates method for applying a mixture of chlorine andchlorine dioxide to a recirculating domestic hot water system fortreatment of biofilms. The physical and chemical parameters in theexample, such as the chemical composition of the treatment solution,temperature of the treatment solution, pH of the treatment solution,flow rates, treatment time, and sequence, are for illustration purposesand are not intended to limit the scope of the invention.

A dosing tap is installed at the output side of the building'scentralized water heater. A chemical feed pump compatible with thetreatment solution is connected to the dosing tap. A sample tap isinstalled at the hot water return. Fixtures (taps, showerheads)throughout the building are prepared by removing aerators andpoint-of-use filters. Off-gas prevention devices—e.g., flexible hosesthat serve as a conduit from the point where the treatment solutionexits the fixture to the drain—are attached to each outlet. Unheateddomestic water is circulated through the hot water distribution systemat 2-8 feet per second (fps).

Chlorine and chlorine dioxide are applied to the circulating water atthe dosing tap such that the resultant composition is a treatmentsolution with a concentration of 50 mg/L (˜25 mg/L each of chlorine andchlorine dioxide) at pH 7.5. Sodium silicate, a complexing agent isapplied to the circulating water to achieve a concentration of 25 mg/L.The treatment solution is circulated through the domestic hot watersystem for one hour. The concentration of the treatment solution ismeasured at the hot water return every 5 minutes; if the concentrationis >5% less than the 50 mg/L set point, additional chemicals are appliedat the dosing tap until the target concentration of the treatmentsolution, measured at the hot water return, is achieved.

Progressing through the facility, starting at the dosing point, taps areopened to full flow until the treatment solution concentration reachesthe 50 mg/L set point; the flow is then reduced to 0.25 gallons perminute (gpm), and the water is allowed to flow for an additional 5minutes, then turned off. Owing to the design of the off-gas containmentdevice, the treatment solution remains in contact with all wettablesurfaces of the tap.

After all the taps have been flushed with the treatment solution andclosed, the treatment solution is circulated through the system foradditional 1 hour. The chemical feed pump is turned off, and the hotwater system is flushed with clear potable water for 30 minutes.

Starting at the dosing point and progressing through the facility, alltaps are opened to full flow and flushed with clean, unheated domesticwater until the concentrations of chemicals in the water are below theEPA Maximum Contaminant Level (MCL) and Maximum Disinfectant ResidualLevel (MRDL) limits, which are the levels to which disinfectants ordisinfection by-products are regulated. Clean water is then allowed toflow through the tap for an additional 5 minutes. The concentration ofthe treatment solution is re-measured and documented to be below theMRDL/MCL for each regulated disinfectant/disinfection by-product. Thetap is turned off and the gas-containment device is removed.

After the acute treatment, the domestic hot water system may be treatedto provide ongoing microbial control.

All publications, patents, patent applications and other documents citedin this application are hereby incorporated by reference in theirentireties for all purposes to the same extent as if each individualpublication, patent, patent application or other document wereindividually indicated to be incorporated by reference for all purposes.Full citations for these references may be found at the end of thespecification immediately preceding the claims.

While various specific embodiments have been illustrated and described,it will be appreciated that various changes can be made withoutdeparting from the spirit and scope of the invention(s).

REFERENCES

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What is claimed is:
 1. A method for eradicating biofilm from a premiseplumbing system, the method comprising contacting the biofilm oninterior surfaces of the premise plumbing system with a treatmentsolution comprising a mixture of chlorine and chlorine dioxide in aratio by weight of from 80:20 to 20:80, each component of the mixturehaving a concentration of at least 1.5 mg/L, wherein the treatmentsolution has a temperature of between 20 to 55° C.
 2. The method ofclaim 1, wherein the treatment solution comprises a mixture of chlorineand chlorine dioxide in a ratio by weight of 50:50.
 3. The method ofclaim 1, wherein the concentration of each of chlorine and chlorinedioxide is at least 3.0 mg/L, at least 6.25 mg/L, at least 12.5 mg/L, orat least 50 mg/L.
 4. The method of claim 1, wherein the treatmentsolution has a pH of about 6.5 to about 9.0.
 5. The method of claim 1,wherein the treatment solution has a pH of between 7.2 and 9.0.
 6. Themethod of claim 1, wherein the treatment solution has a pH of about 7.5.7. The method of claim 1, wherein the treatment solution has a pHof >7.2.
 8. The method of claim 1, wherein the treatment solution has apH of >7.5.
 9. The method of claim 1, wherein the premise plumbingsystem has a free residual concentration of 0.8 mg/L or less chlorinedioxide and 0.4 mg/L or less chlorine.
 10. The method of claim 1,wherein the interior surfaces of the premise plumbing system comprise amaterial selected from the list consisting of copper, brass, iron,galvanized steel, stainless steel, PVC, HDPE, and combinations thereof.11. The method of claim 1, wherein the biofilm comprises a microorganismselected from the group consisting of Acinetobacter, Elizabethkingia(Flavobacterium), Escherichia coli, Klebsiella, Legionella,non-tubercular Mycobacteria (NTM), Pseudomonas, Stenotrophomonas,protozoa, and combinations thereof.
 12. The method of claim 1, whereinthe treatment solution further comprises a complexing agent.
 13. Themethod of claim 12 wherein the complexing agent comprises sodiumsilicate.
 14. The method of claim 1, wherein the said contacting iscarried out under turbulent flow conditions.
 15. The method of claim 14,wherein the turbulent flow conditions-is have a Reynolds value of atleast 4,000.